Gene expression is largely controlled at the level of transcription initiation. The regulation of messenger RNA synthesis is of paramount importance in developmental processes and in oncogenesis. Despite a large body of knowledge about the regulatory factors that contra transcription ,their mechanism of action remains elusive. This is largely due to ignorance about the initiation reaction itself. This project will define the interactions that occur between the promoter DNA of a gene, the general initiation factors, and RNA polymerase II. Only by defining the intermediates in the initiation reaction will it be possible to identify those steps which are subject to regulation. Initially, this work will use a hybrid in vitro transcription system consisting of a mammalian promoter; Transcription Factor (TF)IID and TFIIA from yeast or human cells; and pol II, TFllB, TFllE, and TFllF derived from mammalian cells. This system has already been useful for resolving intermediate transcription complexes by native gel electrophoresis. These studies will be continued to further explore interactions between factors, the transcriptional competence of the complexes and the contribution of promoter sequences to the stability of the complexes. Another important goal of this project will be to develop in vitro trAnscription/native gel electrophoresis system in which all components are derived from the yeast Saccharomyces cerevisiae. Development of this system will complement a concurrent genetic analysis of yeast transcription factors. Specifically, genetic techniques will be used to isolate mutant forms of TFllD and compensating mutation sin other interfacing genes. These interaction sat the genetic level will be explored by reproducing them in the biochemical analysis. The yeast homologues of several cloned mammalian transcription factors will be cloned so that they may also be analyzed by a combined molecular, biochemical, and genetic approach. Ultimately, once the basic initiation reaction is well defined, it will be possible to ask detailed mechanistic questions about how regulatory proteins affect the initiation reaction. THis type of analysis can eventually be applied to the transcription regulatory proteins which are important in oncogenesis (FOS, UN, MYC, etc.). Normal proteins can be compared with oncogenic derivations to determine how they differ in function. Insight into these differences may eventually help design therapeutic treatments.